The detection of foreign substances in body fluids is often essential to the proper diagnosis of a diseased state and selection of the appropriate treatment therefor. The foreign substances, generally termed antigens, may have associated therewith the capacity to stimulate the formation of a corresponding antibody which reacts specifically with that antigen. The antibody itself is a protein that is formed in response to the presence of an antigen for reaction with that specific antigen. Antibodies comprise a special group of serum proteins called immunoglobulins. Although the group of antibodies comprises a restricted group of proteins that are capable of specifically reacting with antigens, there is an enormous variety of macromolecules capable of behaving as antigens, including proteins, many polysaccharides, neucleoproteins, lipoproteins, numerous synthetic polypeptides as well as many other small molecules, called haptens, when they are suitably linked to proteins or synthetic polypeptides.
The specificity of antibody-antigen reactions has been utilized in the diagnosis of pathological states or physiological conditions and more particularly, in the detection of antigenic determinants. As used herein, the phrase "immunological substance" shall be defined as either an antibody or an antigen while the phrase "immunological homolog" shall be defined as the complement of the immunological substance which is capable of specific reaction therewith. Consequently, if the immunological substance being discussed is an antibody, then the immunological homolog would be the antigen for which that antibody is specific. The converse is equally contemplated.
In accordance with the knowledge of those skilled in the art, antigen-antibody reactions can be manifested by enzyme immunoassay, radioimmunoassay, or immunofluorescence techniques with high sensitivity; however, in large part, these techniques are limited to the sensitivity of typically complex instrumentation designed to locate and quantify the marker substances employed.
A well known class of prior art techniques for detecting an antigen-antibody reaction involved labeling the antibody with a tag or marker substance. This technique, however, possesses several disadvantages. The antibody protein is a very sensitive protein whose reactivity, the capability of selectively reacting with its immunological homolog present even in small concentrations, can be easily destroyed by the chemical addition of marker substances to the protein, i.e., denaturation. Further problems include the inability to attach a sufficient concentration of weakly fluorescent but desirable dye molecules such as the red excited fluorescent dye in order to get into a detectable range. On the other hand, if too many dye molecules are attached, then, even though denaturation of the antibody may not occur, nonspecific staining is likely because of the hydrophobic nature of the antibody-dye complex. Additionally, the loss of specificity occasioned by the presence of cationic charges, present on many dyes, makes such a system undesirable.
The concentration at the site of an immunological reaction of a marker substance (and thus the sensitivity of detection) may be increased by employing an indirect staining technique wherein a second immunoglobulin, directed against the first immunoglobulin, carries several dye molecules attached in normal fashion. Since the second immunoglobulin is typically heterospecific, i.e., it binds to several sites, the attachment of several second immunoglobulins to the first is possible. Consequently, the attachment of significantly greater numbers of dye molecules onto the first immunoglobulin, specific against the antigen to be detected, increases the antigen detection sensitivity. Unfortunately, such a procedure involves two reaction steps making it unsuitable for facile use in automated instrumentation. Additionally, while the second antibody system does increase detection sensitivity, it still suffers from the same limitations present in the directly labelled antibody system first described.
It is an object of the present invention to minimize the loss of reactivity and sensitivity due to nonspecific binding occasioned by the above methods.
Another class of well-known prior art techniques employs a biotin-avidin complex which binds by physical adsorption. Although biotin-avidin does not result in a covalent bond, is nonetheless exhibits a very high binding constant. The biotin is, in relation to the antibody, a comparatively small molecule so that the antibody is capable of carrying a number of biotin molecules on its surface. The addition of several labelled avidins to biotinylated antibody results in specific adsorption thereby yielding labelled antibody. Because the avidin is also a small molecule, less than half the size of an antibody, the amount of dye that can be attached to the avidin is limited. It is an object of the present invention to eliminate this limitation and still enjoy the advantages presented by employment of the biotin-avidin technique for linking an antibody to another substance.
Further attempts to increase the sensitivity of immunological reaction detection systems have employed the substitutions of radioisotopes for dyes. Radioisotopes are physically small labels and thereby minimize steric hindrances and sensitivity losses and permit the most sensitive level of detection. The use of radioisotopes, however, presents numerous disadvantages including the relatively short life of gamma emitting isotopes, e.g. .sup.125 I, the impairment of immunological reactivity by gamma radiation of the isotope, health hazards involved in the use of dangerous radioisotopes necessitating the use of procedures complying with federal standards, as well as requiring precise safety controls in addition to expensive, complex detection instrumentation. Further, the radioimmunoassay while suitable for soluble antigens is not readily adaptable on a cell by cell basis since it is really an averaging technique. To make the radioimmunoassay sensitive to a single cell for automated flow cytometry instrumentation would require advanced radiographic techniques. Such techniques would be expected to be slow and procedurally very complex. As a result, radioisotopes are generally nonsuitable for individual cell analysis. It is an object of the present invention to provide a reagent which will increase the sensitivity of autoradiographic techniques and radioimmunoassays and to decrease the possibility of radiation damage normally caused by direct labelling due to close physical proximity of the radioactive label to the antibody.
Enzymes, used in substitution for dye marker substances, advantageously provide an improvement in sensitivity due to the great turnover of substrate. Although such a system requires additional steps beyond the use of the typical dye marker substance, there is no photobleaching problem as evidenced with the use of fluorescent dyes. Furthermore, propitious choice of the enzyme permits the production of a colored compound or an acid readily discerned and quantified by automated equipment. Disadvantages include the large and sticky nature of enzymes which often results in denaturation of the antibody, and a loss of specificity due to the inherent nature of enzymes for nonspecific attachment or steric hindrance from homolog binding. Furthermore, enzymes present shelf life problems which reduce their effectiveness for practical use in clinical environments. If specific structures on the cell are to be localized or measured, then the substrate must precipitate directly on the cell in a conveniently detectable form, e.g., a fluorescent compound. A problem generally encountered with enzyme systems is a limitation in sensitivity because of a fixed and limited enzymatic rate. Compensation can be made for this by simply waiting longer for the enzyme to continue to act upon the substrate. This, of course, reduces the efficiency of such a technique in clinical applications and its usefulness in automated procedures where system throughput capability is of paramount importance. It is an object of the present invention to provide a reagent, compatible with enzymes, for detecting immunological reactions with increased sensitivity.
Electron opaque stains have found useful application in electron microscopy, however, such a system is far more complex and requires lengthy procedures thereby effectively eliminating its clinical value. To some degree, this disadvantage is compensated by an increase in resolution. Electron opaque stains include ferritic compounds, i.e., proteins containing iron or colloidal gold, however, it is noted that these compounds may be larger than the antibody and may therefore be expected to deleteriously affect the antibody's reactivity. It is an object of this invention to reduce such an effect on the antibody as well as to provide a reagent that is electronically dense and opaque to electron scanning.
Traditional attempts to overcome many of the above-described problems include the use of polymer particles or microspheres containing fluorescein or other marker type substances. Generally these polymer particles are constructed in the range of 50-2000 nanometers and consequently, the number that can be attached to a cell through an antibody is sterically limited. Tyically, the particles are coated with large numbers of antibodies; however, due to the physically large nature of the reagent, there is a greatly reduced chance of the antibody approaching an antigenic site on the cell in an appropriate orientation to result in the immunological reaction. Traditionally, the polymer particles have been made from styrenes and vinyl monomers by emulsion polymerization; however, the resulting hydrophobic particles disadvantageously exhibit nonspecific binding as well as colloidal instability. The latter instability is aggravated by the attachment of antibodies on the particle's surface. Of further disadvantage is the surface denaturation affect on the antibody following attachment of the antibody on the surface of the microsphere.
Attempts have been made to overcome the hydrophobic nature of polymeric particles by the incorporation of hydrophilic monomers into the particles, however, these attempts have been limited to emulsion polymerization procedures. Although alteration of the composition of the particles can improve nonspecific binding and colloidal instability problems, these undesirable characteristics cannot be completely eliminated since a minimal amount of hydrophobic monomer is required in order to synthesize the polymeric particle. One such polymeric particle detection system is described in U.S. Pat. No. 4,254,096 to Monthony et al wherein an assay method is described using antibodies covalently bound to hydrophilic polymeric particles which are water-insoluble, an undesirable characteristic. Even in the case of completely hydrophilic monomers, cross-linking agents are needed in order to make a hydrophilic particle as described in U.S. Pat. No. 3,853,987 to Dreyer. It is an object of the present invention to eliminate the requirement for a cross-linking agent.
Hirschfeld and Eaton describe another technique to increase sensitivity using a polymeric backbone in U.S. Pat. No. 4,169,137. They specifically describe an antigen detecting reagent consisting of a primary amine containing, polyfunctional polymeric backbone, i.e., polylysine, having coupled therewith a plurality of fluorescent dye molecules. The polyfunctional backbone was covalently bound through the primary amine on the polylysine to a primary amine moiety on the antibody by the use of a dialdehyde such as glutaraldehyde. Although such a method results in greater sensitivity per antigen site, serious limitations are presented by the described methodology. Specifically, the polymer is restricted to a primary amine containing polymer which, because of its cationic charges, exhibits strong nonspecific binding thereby greatly reducing its specificity. In fact, it was found that the type of polymer described by Hirschfeld attached to cells even without an antibody. Of further disadvantage is the requirement of a dialdehyde coupling reagent. These restrictions serve to drastically limit the advantageous attachment of various fluorescent dyes to the polymeric backbone. Also, the employment of a dialdehyde does not permit the attachment of nonprimary amine containing polymeric backbones to an antibody. When employed within the pH ranges of interest for biological applications, the described system tends to be cationically charged and therefore attaches nonspecifically, by electrostatic interaction, to anionically charged cell and tissue surfaces. Due to the unstable nature of the dialdehyde formed, covalent linkage and the reversible dialdehyde reaction, it is expected that Hirschfeld's reagent will contain much disassociated polymer and antibody capable of competitively reacting with antigenic sites and thereby reducing the sensitivity of the assay.
It is an object of the present invention to provide an immunological substance detection assay employing a water-soluble polymer to carry many detectors, and to avoid nonspecific attachment due to hydrophobic and cationic charge characteristics as well as to avoid the need for dialdehyde linking agents.
It is another object of the present invention to provide a reagent capable of detecting an immunological substance with increased sensitivity. It is a further object to avoid the problems associated with hydrophobic polymers and particles and cationically charged complexes which disadvantageously result in nonspecific binding by providing properly charged polymers having minimum hydrophobic characteristics. It is an object to provide a water-soluble polymer, capable of carrying a plurality of marker substances, which does not have a net positive charge.
It is yet another object to provide a reagent capable of simple storage and effective use in automated instrumentation. It is a still further object to provide a reagent capable of employing the desirable, but weakly fluorescent, red excited fluorescent dyes in concentrations and amounts capable of being detected with the technology presently employed in automated cytology instrumentation.
It is a yet further object to employ a water-soluble polymer in combination with avidin-biotin in order to utilize the individual advantages of each within a single reagent.